Each reference cited herein is expressly incorporated herein by reference in its entirety.
Diabetes mellitus is one of the most common chronic diseases worldwide characterized by progressive loss of functional insulin-producing β-cells, resulting in hyperglycemia associated with diabetic complications. It has been predicted that over 300 million people worldwide will be diagnosed with type I or type II diabetes by the year 2025 (Zimmet, Paul, K. G. M. M. Alberti, and Jonathan Shaw. “Global and societal implications of the diabetes epidemic.” Nature 414, no. 6865 (2001): 782-787.). These diseases induce other diseases, such as heart disease and stroke, high blood pressure, kidney disease, and blindness. Type 1 diabetes results from autoimmune destruction of β-cells, leading to incapable of maintaining normoglycemia in these patients. Although type 2 diabetes is caused by the peripheral resistance to insulin and impaired insulin secretion, the late stages of this disease can induce a significant decrease in β-cell mass. Therefore, both type 1 and type 2 diabetic patients could benefit from β-cell replacement therapy. Islet transplantation has proven to be a cure to diabetes. Nevertheless, this treatment is unavailable to vast majority of patients due to the scarcity of human donors and transplant rejection. Thus, new sources of transplantable islets need to be identified.
While islet transplantation is promising, the supply of transplantable islet tissues remains a challenge. Great efforts have been made to generate biologically functional islet-like organoids from human pluripotent stem cells (hPSCs) for diabetes treatment.
Human pluripotent stem cells (hPSCs) have a great potential to become a major cell source to produce biologically functional insulin secreting β-cells for cell-based therapy. In spite of intense efforts made to enhance hPSC β-cell differentiation over the past decade, current approaches focus on generating insulin secreting β-cells, rather than islets or islet organoids. This is due to the fact that niches inducing in vitro self-assembly of islet organoids from hPSCs have NOT to be identified yet.
The pancreas arises from both dorsal and ventral portions of foregut endoderm [Y. Wen, S. Jin, Production of neural stem cells from human pluripotent stem cells, J Biotechnol 188 (2014) 122-9]. The formation of a multipotent pancreatic epithelium after a rapid growth of pancreatic buds from the foregut endoderm leads to the development of both pancreatic exocrine and endocrine structures that work intimately to regulate nutrient metabolism and blood glucose concentration. Distinct endocrine cells, including insulin (INS)-producing β cells, glucagon (GCG)-producing α cells, somatostatin (SST)-producing δ cells, pancreatic polypeptide (PP)-producing PP cells, and ghrelin-producing ε cells, are organized into cell clusters forming the islets of Langerhans that are embedded in the glandular exocrine pancreas and are closely associated with microvasculature and neurovascular environments [S. Jin, H. Yao, P. Krisanarungson, A. Haukas, K. Ye, Porous membrane substrates offer better niches to enhance the Wnt signaling and promote human embryonic stem cell growth and differentiation, Tissue Eng Part A 18(13-14) (2012) 1419-30]. These development processes are highly regulated and controlled by many factors such as morphogens and key transcriptional regulators produced by surrounding embryonic development environments.
Most current knowledge about pancreogenesis during embryo development have been gleaned from studies using rodent models. Nevertheless, a line of evidence suggests significantly different developmental events occurred during human pancreogenesis as compared to those observed in mice [S. Jin, H. Yao, J. L. Weber, Z. K. Melkoumian, K. Ye, A synthetic, xeno-free peptide surface for expansion and directed differentiation of human induced pluripotent stem cells, PLoS One 7(11) (2012) e50880; Y. Stefan, L. Orci, F. Malaisse-Lagae, A. Perrelet, Y. Patel, R. H. Unger, Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans, Diabetes 31(8 Pt 1) (1982) 694-700; H. Ichii, L. Inverardi, A. Pileggi, R. D. Molano, O. Cabrera, A. Caicedo, S. Messinger, Y. Kuroda, P. O. Berggren, C. Ricordi, A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations, Am J Transplant 5(7) (2005) 1635-45; M. Brissova, M. J. Fowler, W. E. Nicholson, A. Chu, B. Hirshberg, D. M. Harlan, A. C. Powers, Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy, J Histochem Cytochem 53(9) (2005) 1087-97; T. A. Matsuoka, I. Artner, E. Henderson, A. Means, M. Sander, R. Stein, The MafA transcription factor appears to be responsible for tissue-specific expression of insulin, Proc Natl Acad Sci USA 101(9) (2004) 2930-3]. The distinct cytostructure between human and mouse islets further implies different developmental mechanisms adopted by the two species. In mice, β cells form a core of the islets, while such a core structure does not exist in human islets. In human islets, β, α, δ, PP, and ε cells are mixed and connected with blood vessels, nerve fibers, and lymphatic vessels [C. Zhang, T. Moriguchi, M. Kajihara, R. Esaki, A. Harada, H. Shimohata, H. Oishi, M. Hamada, N. Morito, K. Hasegawa, T. Kudo, J. D. Engel, M. Yamamoto, S. Takahashi, MafA is a key regulator of glucose-stimulated insulin secretion, Mol Cell Biol 25(12) (2005) 4969-76; Pagliuca, Felicia W., Jeffrey R. Millman, Mads Gürtler, Michael Segel, Alana Van Dervort, Jennifer Hyoje Ryu, Quinn P. Peterson, Dale Greiner, and Douglas A. Melton. “Generation of functional human pancreatic β cells in vitro.” Cell 159, no. 2 (2014): 428-439; X. Wang, K. Ye, Three-dimensional differentiation of embryonic stem cells into islet-like insulin-producing clusters, Tissue Eng Part A 15(8) (2009) 1941-52]. Consequently, differentiation protocols developed based on mechanisms elucidated from animal studies might not be sufficient to generating biologically functional β cells for cell-base diabetes treatment.
The generation of islet organoids has been attempted in the last decades. In previous work, the feasibility of assembly of islet-like cell clusters from pancreatic differentiated mouse embryonic stem cells (mESCs) within a collagen scaffold was demonstrated [Wang X, Ye K. Three-dimensional differentiation of embryonic stem cells into islet-like insulin-producing clusters. Tissue Eng Part A 15, 1941-1952 (2009)]. mESC-derived cell clusters were produced which consisted of α, β, and δ cells. They exhibited a characteristic mouse islet architecture that has a β cell core surrounded by a and 6 cells. The islet-like cell clusters produced ATP-sensitive K+ (KATP) channel dependent insulin secretion upon glucose challenging. However, no PP cells were detected in these cell clusters, suggesting that these cell clusters are distinct to adult islets. Built upon these findings, the generation of islet organoids (consisting all endocrine cells) from human embryonic stem cells (human ESCs, or hESCs) within a biomimetic scaffold was demonstrated [Wang W, Jin S, Ye K. Development of Islet Organoids from H9 Human Embryonic Stem Cells in Biomimetic 3D Scaffolds. Stem Cells Dev, (2016)]. The cytostructural analysis of these organoids revealed a typical architecture of human adult islets. These organoids consisted of α, β, δ, and PP cells. Both β cells and non-β cells were mixed randomly to form organoids that secrete insulin in response to glucose challenges.
A recent study reported by Kim et. al. suggested the therapeutic effects of ESC-derived islet-like organoids in a diabetic mouse model [Kim, Youngjin, Hyeongseok Kim, Ung Hyun Ko, Youjin Oh, Ajin Lim, Jong-Woo Sohn, Jennifer H. Shin, Hail Kim, and Yong-Mahn Han. “Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo.” Scientific reports 6 (2016): 35145]. Their animal studies suggested the possibility of suppressing hyperglycemia in Streptozotocin (STZ)-induced diabetic mice after islet-like organoid transplantation. However, the normalization of blood glucose level in these transplanted mice only lasted 40 days. The long-term therapeutic benefits of these cell clusters has not been demonstrated. The analysis of the cytostructure of these islet-like organoids revealed insufficient endocrine cell composition. No a cells and only very few δ cells were detected in these cell clusters. The detection of pancreatic endocrine marker gene expression indicated a low level of expression of Nkx6.1, a mature gene marker of β cells in these islet-like organoids. The expression of MAFB was also detected in these cell clusters, further suggesting their immaturity.
The study reported by Pagliuca et al. represented a success in generating functional human pancreatic β cells from human pluripotent stem cells (HPSCs) in a suspension culture [Pagliuca F W, et al. Generation of functional human pancreatic beta cells in vitro. Cell 159, 428-439 (2014)]. The transplantation of these insulin-secreting cells in diabetic mice led to long-term glycemic control, suggesting potential use of these cells for diabetes therapy [Vegas A J, et al. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nat Med 22, 306-311 (2016)]. In their study, they discovered that cell clusters were formed after culturing hPSC-derived INS+/NKX6.1+ cells in a suspension culture. The size of these cell clusters was around ˜200 μm that is larger than human islets (˜100 μm). No PP cells were detected in these cell clusters. Only minor populations of α and δ cells were found in these cell clusters, as compared to adult islets which contains roughly 20% α-cells, 10% δ cells, and <5% PP cells [Stefan Y, Orci L, Malaisse-Lagae F, Perrelet A, Patel Y, Unger R H. Quantitation of endocrine cell content in the pancreas of nondiabetic and diabetic humans. Diabetes 31, 694-700 (1982); Ichii H, et al. A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations. Am J Transplant 5, 1635-1645 (2005); Brissova M, et al. Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy. J Histochem Cytochem 53, 1087-1097 (2005)]. The population of both α and δ cells in cell clusters increased after grafting, suggesting the further maturation of these cell clusters in vivo.
The generation of islet-like cell clusters from either human ESCs or iPSCs has been investigated in a number of studies including the use of miR-186 and miR-375 to enhance differentiation of iPSCs into islet-like cell clusters [Wang X, Ye K. Three-dimensional differentiation of embryonic stem cells into islet-like insulin-producing clusters. Tissue Eng Part A 15, 1941-1952 (2009); Jiang J, et al. Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells 25, 1940-1953 (2007); Tateishi K, He J, Taranova O, Liang G, D'Alessio A C, Zhang Y. Generation of insulin-secreting islet-like clusters from human skin fibroblasts. J Biol Chem 283, 31601-31607 (2008); Shaer A, Azarpira N, Karimi M H. Differentiation of human induced pluripotent stem cells into insulin-like cell clusters with miR-186 and miR-375 by using chemical transfection. Appl Biochem Biotechnol 174, 242-258 (2014); Shaer A, Azarpira N, Vandati A, Karimi M H, Shariati M. Differentiation of human-induced pluripotent stem cells into insulin-producing clusters. Exp Clin Transplant 13, 68-75 (2015); Ionescu-Tirgoviste C, et al. A 3D map of the islet routes throughout the healthy human pancreas. Sci Rep 5, 14634 (2015); Rezania A, et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133 (2014)]. In light of these successes, cytostructure and endocrine cell compositions of these cell clusters are distinct markedly from adult islets. In particular, no PP cells are detected from these cell clusters. Most endocrine cells in these cell clusters express multiple hormones such as INS and GCG, similar to marker gene expression patterns detected in pancreatic endocrine progenitors. Shim, et. al. showed that the INS+/GCG+ cells disappeared after cell cluster grafting in STZ-induced diabetic mice, presumptively due to in vivo maturation of these cell clusters [Shim J H, et al. Pancreatic Islet-Like Three-Dimensional Aggregates Derived From Human Embryonic Stem Cells Ameliorate Hyperglycemia in Streptozotocin-Induced Diabetic Mice. Cell Transplant 24, 2155-2168 (2015)]. The generation of islet-like cell clusters from human umbilical cord mesenchymal stem cells has also been explored [Chao K C, Chao K F, Fu Y S, Liu S H. Islet-like clusters derived from mesenchymal stem cells in Wharton's Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS One 3, e1451 (2008)].
In addition, extensive efforts have made to generate glucose-responsive, insulin-secreting β cells in last two decades [Pagliuca F W, et al. Generation of functional human pancreatic beta cells in vitro. Cell 159, 428-439 (2014); Rezania A, et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133 (2014); Russ H A, et al. Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro. EMBO J 34, 1759-1772 (2015); Takeuchi H, Nakatsuji N, Suemori H. Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 4, 4488 (2014); Rajaei B, Shamsara M, Massumi M, Sanati M H. Pancreatic Endoderm-Derived from Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells. J Cell Physiol, (2016); Fotino N, Fotino C, Pileggi A. Re-engineering islet cell transplantation. Pharmacol Res 98, 76-85 (2015); D'Amour K A, et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24, 1392-1401 (2006); Rezania A, et al. Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo. Stem Cells 31, 2432-2442 (2013); Jiang W, et al. In vitro derivation of functional insulin-producing cells from human embryonic stem cells. Cell Res 17, 333-344 (2007); Zhu S, et al. Human pancreatic beta-like cells converted from fibroblasts. Nat Commun 7, 10080 (2016)]. Growing evidences suggest that islet structure is critical to the maturation of β cells during pancreatic organogenesis [Li Y, Xu C, Ma T. In vitro organogenesis from pluripotent stem cells. Organogenesis 10, 159-163 (2014)]. The heterotypic contact between α and β cells in human islets suggests paramount role of a cells to β cells during their maturation and glucose-responsive insulin-secretion [Nair G, Hebrok M. Islet formation in mice and men: lessons for the generation of functional insulin-producing beta-cells from human pluripotent stem cells. Curr Opin Genet Dev 32, 171-180 (2015); Halban P A. Cellular sources of new pancreatic beta cells and therapeutic implications for regenerative medicine. Nat Cell Biol 6, 1021-1025 (2004); Rorsman P, Braun M. Regulation of insulin secretion in human pancreatic islets. Annu Rev Physiol 75, 155-179 (2013)]. The formation of gap junctions during cell-cell coupling has been found crucial to functional mature of β cells [Carvalho C P, et al. Beta cell coupling and connexin expression change during the functional maturation of rat pancreatic islets. Diabetologia 53, 1428-1437 (2010); Benninger R K, Piston D W. Cellular communication and heterogeneity in pancreatic islet insulin secretion dynamics. Trends Endocrinol Metab 25, 399-406 (2014)]. Accordingly, the composition and relative proportion of islet cells have profound effect in regulating pancreatic endocrine cell maturation and their physiological functions in vivo, which necessitates the formation of islets or islet organoids consisting of all islet cell types.
See, U.S. Pat. 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Youngjin Kim, Hyeongseok Kim, Ung Hyun Ko, Youjin Oh, Ajin Lim, Jong-Woo Sohn, Jennifer H. Shin, Hail Kim, and Yong-Mahn Han, “Islet-like organoids derived from human pluripotent stem cells efficiently function in the glucose responsiveness in vitro and in vivo”, Sci Rep. 2016; 6: 35145, 2016 Oct. 12, doi: 10.1038/srep35145, PMCID: PMC5059670, reports that insulin secretion is elaborately modulated in pancreatic β cells within islets of three-dimensional (3D) structures. Using human pluripotent stem cells (hPSCs) to develop islet-like structures with insulin-producing β cells for the treatment of diabetes is challenging. Pancreatic islet-like clusters derived from hESCs are functionally capable of glucose-responsive insulin secretion as well as therapeutic effects. Pancreatic hormone-expressing endocrine cells (ECs) were differentiated from hESCs using a step-wise protocol. The hESC-derived ECs expressed pancreatic endocrine hormones, such as insulin, somatostatin, and pancreatic polypeptide. Notably, dissociated ECs autonomously aggregated to form islet-like, 3D structures of consistent sizes (100-150 μm in diameter). These EC clusters (ECCs) enhanced insulin secretion in response to glucose stimulus and potassium channel inhibition in vitro. Furthermore, β cell-deficient mice transplanted with ECCs survived for more than 40 d while retaining a normal blood glucose level to some extent. The expression of pancreatic endocrine hormones was observed in tissues transplanted with ECCs. In addition, ECCs could be generated from human induced pluripotent stem cells. These results suggest that hPSC-derived, islet-like clusters may be alternative therapeutic cell sources for treating diabetes.
US 2007/0037281, expressly incorporated herein by reference in its entirety, discusses a method for differentiating stem cells in cells that produce a pancreatic hormone. See, WO02/059278.
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